The field of this invention relates to the stabilization of dried bacteria against loss of viability during non-refrigerated storage prior to use. More particularly, the invention is concerned with the stabilization of dried bacteria dispersed in particulate carriers.
Living bacteria have heretofore been combined with particulate carriers to provide admixtures which may be stabilized and stored. See, for example, U.S. Pat. Nos. 3,677,898 (Jeffreys), 3,898,132 (Hettrick), and 4,229,544 (Haynes, et al). Jeffreys describes the coating of lactic acid bacteria with an acetylated monoglyceride, and the combining of the coated bacteria with a starch or sugar carrier mixture which may contain minor amounts of inorganic salts. Hettrick mixes bacteria in aqueous dispersions with diatomaceous earth particles to produce a storable dormant mixture. Haynes, et al. combines bacteria with a carrier such as peat prior to packaging of the bacteria. Particulate desicants have been proposed for use in the freeze-drying of bacteria, as disclosed in U.S. Pat. Nos. 2,853,797 and 2,897,600, but not for admixture with carrier diluents.
Other relevant prior art includes U.S. Pat. Nos. 4,205,132 (Sandine, et al.), 4,518,696 (Gehrman, et al.), 4,115,199 (Porubcan, et al.), 3,897,307 (Porubcan, et al.), 3,616,236 (Delin), 1,957,555 (Reichel et al.), and Hunt et al., "Preservation of Cultures by Drying on Porcelin Beads", J. of Bacteriology, Vol. 76, pp. 453-454, 1958, De Silva, et al. "Preservation of Lactic Acid Bacteria on Anhydrous Silica Gel for Three Years", J. Food Protection, Vol. 46, pp. 699-701, August, 1983, Trollope, "The Preservation of Bacteria and Fungi on Anhydrous Silica Gel: an Assessment of Survival Over Four Years", J. appl. Bact., Vol. 38, pp. 115-120, 1975.
Sandine et al. describes a method of stabilizing freeze-dried viable bacteria, such as lactic acid producing bacteria in combination with inorganic buffering salts which increase the rate of survival of the bacteria. The bacteria is mixed with a small amount of an alkali metal salt of glycerophosphate in an aqueous medium including milk solids with a pH between 6 and 8. The mixture is frozen and vacuum sealed with the introduction of argon to provide an essentially oxygen-free environment. About 10% of the bacteria surviving lyophilization remain viable for 90 days.
Gehrman et al. describes a stabilized liquid bacterial composition for use as a drench for administration to animals. Porubcan '307 describes the use of dried cultures of lactic acid producing bacterial cells combined with ascorbates, glutamate or aspartate.
De Silva et al. suggests that lactic acid producing bacteria may be effectively stored for long periods of time when in combination with silica gel particles which act as desiccants at low temperatures. Cultures can be stored at 5.degree. C. for four years.
Trollope reports that bacteria tend to survive preservation on silica gel better after drying or lyophilization. Delin describes the procedure for drying mixed Rhizobium strains of nitrogen-fixing bacteria to destroy the weaker strains. A variety of different drying procedures are described. These include the use of desiccants and various heat drying procedures. The drying procedures are to be carried out at a high temperature so that bacteria are killed. The reference states: "Preferably, at least 90% of the bacteria are killed in the course of each drying treatment."
Reichel et al. describes the preparation of neutralized concentrates of Lactobacilli. Neutralizing/buffering agents are added to the moist bacterial concentrates. Preferred additives are milk and milk of magnesia. The resulting product is "a thick viscous liquid which contains a Lactobacilli in the form of a stable concentrate."
Hunt et al. describes a method of preserving bacterial cultures by during on porcelain beads. A liquid broth culture is applied to the beads and thereafter subjected to a drying atmosphere. The atmosphere (not the beads) is in contact with a drying agent, such as silica gel. The bacteria do not contact the silica gel. The reference teaches that such contact is undesirable, stating that the porcelain beads are used "to prevent the contact of the cells with the silica gel which we have found to be deleterious."
As illustrated by some of the foregoing disclosures, the bacteria being stabilized may be intended for a variety of uses in which dilution or extension of a bacterial concentrate is advantageous. The dry bacteria mixtures may be useful in making fermented dairy products, or they may comprise rumen bacteria for feeding to cattle or sheep. They may also be soil bacteria for addition to agricultural soils, or for other purposes.
An important use has developed for Lactobacillus plantarum in producing silage fermentation. Lactobacillus acidophilus is being extensively used with domestic animals to improve their health and well being. In preparing the Lactobacillus for inoculation of silage, and/or for feeding to domestic animals, it is convenient to have the bacteria in highly diluted form, viz. extended in a particulate carrier which is in dry free-flowing condition. But the diluted bacteria must maintain a high degree of stability under non-refrigerated storage conditions for a number of months prior to use. Heretofore the stabilities of lactic acid bacteria in such dispersions have depended on the dry carrier used and the packaging. Results have often been inconsistent with the lactic acid bacteria showing good stability in some dry carriers and poor stability in others. Unpredictable results have been obtained where the same dry carrier that showed good stability at one time later produced poor stability.
No satisfactory method has been known for producing consistent, reproducible stabilization of bacteria in dry carriers. From a practical standpoint, the problem has been further complicated by observations that greater instability, and greater uncertainty of results occur as the degree of dilution of the bacteria is increased. Yet this is exactly the type of formulation most desired for the large scale uses like those of L. plantarum and acidophilus referred to above. Prior art disclosures describe the survivability of lactic acid bacteria at low or room temperature. However, the amount of bacteria surviving extended storage periods is relatively small, especially at room temperature. None of the prior art disclosures previously described disclose methods to stabilize bacteria for extended periods of time while maintaining the viability of the bacteria, i.e., large number of the bacteria to function as inoculates of animal feed.
Concentrated cultures of lactic acid producing bacteria may be prepared by the method of U.S. Pat. No. 4,115,199. Tripolyphosphate and/or hexametaphosphate are added to the culture medium prior to separation of the cells by centrifugation. The resulting concentrates have usually been frozen with liquid nitrogen for use in manufacturing cheese or other dairy products. However, sufficient stabilization for distribution in a dry non-refrigerated form can be obtained by the method described in U.S. Pat. No. 3,897,307. The cell culture is adjusted to a pH favorable to the stability of the cells on drying, and chemical stabilizers are added comprising an ascorbate compound together with either a glutamate compound or an aspartate compound (viz. ascorbic acid with monosodium glutamate). The bacteria are then dried by a suitable procedure; viz. freeze-drying, spray drying or fluid bed drying. Drying to a low moisture content, such as 2.5 to 3.5% by weight, is desirable. Good stability is obtained when the product is packaged in moisture impervious containers. This permits non-refrigerated storage and distribution for uses such as home manufacture of yogurt. Such stabilization procedures, however, have not been adequate where the bacteria are mixed with relatively large amounts of particulate carriers to form highly extended bacterial admixtures, such as for addition to animal feeds or addition to silage materials.